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CROATICA CHEMICA ACTA CCACAA 79 (4) 545¿552 (2006)

ISSN-0011-1643

CCA- 3123

Original Scientific Paper

Chemical Composition and Antioxidant Activity of Essential Oils of Twelve Spice Plants

Olivera Politeo,* Mila Juki}, and Mladen Milo{

Faculty of Chemical Technology, Department of Biochemistry and Food Chemistry, University of Split, Teslina 10/V, 21000 Split, Croatia

RECEIVED AUGUST 31, 2005; REVISED MARCH 2, 2006; ACCEPTED MARCH 16, 2006

Keywords

Chemical compositions and related total antioxidant capacities of twelve spice essential oils were analyzed. To enable a comparison of their relative antioxidant potentials, essential oils were extracted by hydrodistillation from selected spice plants and their chemical compositions were determined by the GC-MS system on two fused-silica capillary columns of different po- larity. Antioxidant effectiveness was examined by four different methods: the 2,2'-diphen- yl-1-picrylhydrazyl (DPPH) radical scavenging method, determination of ferric reducing anti- oxidant power (FRAP), determination of antioxidant activity with thiobarbituric acid reactive species (TBARS) and automatic determination of the oxidative stability of fat (RANCIMAT). Based on their antioxidant capacity, twelve spice essential oils can be sorted in descending or-

spice plants

der: Clove ( Syzygium aromaticum L.) > Basil ( Ocimum basilicum L.) > Laurel ( Laurus nobilis

essential oils

L.) > Coriander ( Coriandrum sativum L.) > Nutmeg ( Myristica fragrans Houtt.) > Black Pep -

chemical composition

per ( Piper nigrum L.) > Everlast ( Helichrysum italicum G. (Roth) Don) > Mint ( Mentha pi -

GC-MS

perita L.) > Marjoram ( Marjorana hortensis Moench.) > Cinnamon ( Cinnamomum zeylanicum

antioxidant activity

Nees) > Sage ( Salvia officinalis L.) > Fennel ( Foeniculum vulgare Muller).

INTRODUCTION

About ten years ago, Aruoma 1 and then Halliwell 2 de- scribed the experimental strategies for optimization of nu- tritional antioxidant intake in humans. The antioxidant pro- perties of many aromatic herbs are reported to be effective in this role. 35 Apart from their use as aroma additives in food, essential oils from aromatic spice plants have a po- tential to be used in small amounts in fat-containing food systems to prevent or delay some chemical deteriorations occurring during the storage of these products. Antioxidant activities of aroma extracts obtained from spices have been investigated in various model sy -

stems. 68 Shahidi et al. 9 reported that the antioxidant ef- fect of aromatic plants is due to the presence of hydroxyl groups in their phenolic compounds. Lagouri et al. 10 stu - died the antioxidant activity of essential oils and they found that oregano essential oil, rich in thymol and car - vacrol, has a considerable antioxidant effect on the process of lard oxidation. In our previous works, 1113 all »pheno- lic« type essential oils, containing thymol and carvacrol as major components, exhibited strong antioxidant activity.

As a part of an investigation of natural antioxidants from spice plants, we report in this paper a study of the antioxidant activities associated with the chemical com -

* Author to whom correspondence should be addressed. (E-mail: olivera @ ktf-split.hr)

546

position of essential oils without significant amounts of thymol and carvacrol, isolated from twelve different spi - ce plants. Our aim is to find out if they can be potent an - tioxidants like the »phenolic« type essential oils descri - bed above and to estimate which of their constituents could be active in this role. For this purpose, the screening of antioxidant power was performed in vitro by four different methods: the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scaveng - ing method, determination of ferric reducing antioxidant power (FRAP), determination of antioxidant activity with thiobarbituric acid reactive species (TBARS) and automatic determination of the oxidative stability of fat (RANCIMAT).

EXPERIMENTAL

Plant Material

Twelve spices: Clove, Syzygium aromaticum L. (Myr - taceae); Basil, Ocimum basilicum L. (Lamiaceae); Laurel, Laurus nobilis L. (Lauraceae); Coriander, Coriandrum sa - tivum L. (Apiaceae); Nutmeg, Myristica fragrans Houtt. (Myristicaceae); Black Pepper, Piper nigrum L. (Pipera- ceae); Everlast, Helichrysum italicum G. (Roth) Don (Com- positae); Mint, Mentha piperita L. (Lamiaceae); Marjoram, Marjorana hortensis Moench. (Lamiaceae); Cinnamon, Cinnamomum zeylanicum Nees (Lauraceae); Sage, Salvia officinalis L. (Lamiaceae) and Fennel, Foeniculum vulgare Muller (Apiaceae) were purchased from a local market in Split, Croatia. Plant materials consisted of flower buds (clo- ve), leaves (basil, laurel, mint, marjoram, sage), fruits (cori- ander, nutmeg, black pepper, fennel), stem bark (cinnamon) and flowered tops (everlast). Voucher specimens of spice plant materials are deposited in the Department of Bioche - mistry and Food Chemistry, Faculty of Chemical Technol - ogy, Split, Croatia.

Isolation of Essential Oils

A hundred grams of dried plant material was subjected to three-hours of hydrodistillation using a Clevenger-type ap - paratus. The obtained essential oils were dried over anhy - drous sodium sulphate and stored under nitrogen in sealed vials at –18 °C until required. The chemicals and all applied solvents were of pro analysis purity and were purchased from Fluka Chemie, Buchs, Switzerland.

Gas Chromatography-Mass Spectrometry

Analyses of volatile compounds were run on a Hewlett – Packard GC-MS system (GC 5890 series II; MSD 5971A, Hewlett Packard, Vienna, Austria). Two columns of differ - ent polarity were used: a HP-101 column (Methyl silicone fluid, Hewlett Packard; 25 m ´ 0.2 mm i.d., film thickness 0.2 m m) and a HP-20M column (Carbowax, Hewlett Packard; 50 m ´ 0.2 mm i.d., film thickness 0.2 m m). Oven

Croat. Chem. Acta 79 (4) 545¿552 (2006)

O. POLITEO et al.

temperature was programmed as follows: isothermal at 70 °C for 4 min, then increased to 180 °C, at a rate of 4 °C min 1 and subsequently held isothermal for 15 min (for HP-20M column); isothermal at 70 °C for 2 min, then in - creased to 200 °C, at a rate of 3 °C min 1 and held isother - mal for 15 min (for HP-101 column). The carrier gas was helium (1 mL/min). The injection port temperature was 250 °C and the detector temperature was 280 °C. Ionization of sample components was performed in the EI mode (70 eV). A volume of 1 m L was injected.

The linear retention indices for all compounds were de - termined by co-injection of the sample with a solution con - taining a homologous series of C 8 -C 22 n -alkanes. 14 The in - dividual constituents were identified by their retention indi - ces identical to the compounds known from literature data, 15 and also by comparing their mass spectra with spec - tra of either the known compounds or with those stored in the Wiley mass spectral database (Hewlett Packard, Vienna, Austria).

Choice of the Method for Determination of Antioxi - dant Activities

As previously described, antioxidant activity assessment re - quires use of different methods. 16,17 Like in numerous stud- ies, 8,1823 DPPH, FRAP, TBARS and RANCIMAT can be cited as relatively simple methods that can be used to mea- sure the antioxidant potential of essential oils. The DPPH method is sensitive and requires little sample material. 24 The TBARS method is also sensitive and achieves repro- ducible results. The FRAP method is fast, easy to handle, with highly reproducible results. 25 Although the RANCI- MAT technique has been questioned, 26 this procedure is commonly used in the food industry and governmental ana- lytical laboratories. 27

Determination of Antioxidant Activity with the 2,2'-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Method

The antioxidant activity of volatile compounds was measu - red in terms of hydrogen donating or radical scavenging ability, using the stable radical DPPH. 28 A methanolic stock solution (50 m L) of the essential oils (concentrations of stock solutions were 50, 20, 10 and 5 g/L) was put into a cuvette, and 2 mL of 6 ´ 10 5 mol L 1 methanolic solution of DPPH was added. Absorbance measurements commenc - ed immediately. The decrease in absorbance at 517 nm was determined with a Perkin-Elmer spectrophotometer after 1 h for all samples. Methanol was used to zero the spectro - photometer. Absorbance of the DPPH radical without anti - oxidant, i.e. the control, was measured daily. Special care was taken to minimize the loss of free radical activity of the DPPH radical stock solution. 24 Percent inhibition of the DPPH radical by the samples was calculated according to the formula of Yen & Duh: 29

% inhibition = (( A C(o) A A(t) ) / A C(o) ) ´ 100

ANTIOXIDANT ACTIVITY OF SPICE ESSENTIAL OILS

where A C(o) is the absorbance of the control a t t = 0 min and A A(t) is the absorbance of the antioxidant a t t = 1 h.

Determination of Ferric Reducing Antioxidant Power (FRAP Assay)

The total antioxidant potential of a sample was determined using the ferric reducing ability of plasma (FRAP) assay of Benzie and Strain 30 as a measure of »antioxidant power«. The FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe II -tripyridyl - triazine compound from the colorless oxidized Fe III form by the action of electron donating antioxidants. Standard curve was prepared using different concentrations (100–1000 m mol/L) of FeSO 4 7H 2 O. All solutions were used on the day of preparation. In the FRAP assay, the antioxidant effi - ciency of the antioxidant tested was calculated with referen- ce to the reaction signal given by an Fe 2+ solution of known concentration, this representing a one-electron exchange re - action. The results were corrected for dilution and expressed in m mol Fe II /L. The sample to be analyzed was first ade - quately diluted to fit within the linearity range.

Determination of Antioxidant Activity with Thiobarbituric Acid Reactive Species (TBARS As- say)

Modified thiobarbituric acid reactive species (TBARS) as- say 20 was used to measure the potential antioxidant capac- ity using egg yolk homogenates as lipid rich media. Briefly, 0.5 mL of 10 % (w/v) homogenate and 0.1 mL of sample solutions to be tested were added to a test tube and made up to 1.0 mL with distilled water. 0.05 mL of 2,2'-azobis (2- amidinopropane) dihydrochloride solution (0.07 mol L 1 )

in water was added to induce lipid peroxidation. 1.5 mL of

20 % acetic acid (pH = 3.5) and 1.5 mL 0.8 % (w/v) thio - barbituric acid in 1.1 % (w/v) sodium dodecyl sulphate so -

lution was added and the resulting mixture was vortexed,

and then heated at 95 °C for 60 min. After cooling, 5.0 mL

of butan-1-ol was added to each tube, then extensively vor -

texed and centrifuged at 1200 g for 10 min. Absorbance of the organic upper layer was measured using a spectropho - tometer (PerkinElmer Lambda EZ 201, Roma, Italia) set at 532 nm. All the values were based on the percentage anti - oxidant index (AI %):

AI % = (1 – A T / A C ) ´ 100

where A C is the absorbance value of the fully oxidized con - trol and A T is the absorbance of the test sample.

Determination of Oxidative Stability of Fat (RANCIMAT)

A Rancimat 743 (Metrohm, Switzerland) was used to deter -

mine the antioxidant lipid activity of volatile compounds contained in the essential oils of the spice plants. The Ran - cimat worked on the following principle: A solution of dif- ferent concentrations of antioxidant (100 m L) was added to

547

the lard (2.5 g) giving a final concentration of 0.20 %, 0.08 %, 0.04 % or 0.02 % of antioxidant in the reacting system. The lard with and without addition of antioxidant was heated at 110 °C and an airflow of 20 L/h was constantly blown into the mixture. The antioxidant activity index (AAI) was calculated from the measured induction times, according to the follo - wing formula by Forster et al . 31

AAI = Induction time of lard with antioxidant / Induction time of pure lard

RESULTS AND DISCUSSION

Chemical Composition of Essential Oils

The analyses were successful without previous fraction - ation of essential oils. Except for laurel (93.0 %), more than 95 percent of constituents were identified in all other essential oil samples. The results of these analyses are presented in Table I as a relative peak area of each constituent. It seems that there were no similarities among chemical compositions of the studied essential oils. Some oils have very simple chemical composition. For example, the clove, coriander and fennel essential oils were composed of only five, eight and seven com- pounds, respectively. On the other side, some oils were very complex. The everlast, nutmeg and, laurel essential oils were composed of 37, 24 and 22 compounds, respe- ctively. Other essential oils had fewer than 20 identified compounds. In some of the essential oils, the main con- stituents accounted for more than 90 % of total oil, e.g., cinnamon ( trans- cinnamaldehyde 94.0 %), coriander (li- nalool 92.0 %) and clove oils (eugenol 91.2 %). In fen - nel essential oil, the content of trans- anethol was 77.6 %; in black pepper, the content of caryophyllene was 57.6 %, and in sage essential oil, the content of thujone was 56.5 %. In other essential oils, the main compounds accounted for less than 50 % of total oil. The main com - pounds of these last ones were the following: estragole (24.7 %) and linalool (23.5 %) in basil oil; neomenthol (44.1 %) and isomenthone (30.9 %) in mint oil; 1.8-ci - neole (34.9 %) and linalool (13.5 %) in laurel oil; ter - pinen-4-ol (40.8 %), g -terpinene (16.3 %) and a -terpine - ne (11.0 %) in marjoram oil; a -cedrene (18.3 %), a -pi - nene (11.3 %) and 2-methylcyclohexyl-pentanoate (10.5 %) in everlast oil, and sabinene (25.4 %), a -pinene (15.8 %), myristicine (14.8 %) and b -pinene (13.4 %) in nut - meg oil.

Antioxidant Activity of Essential Oils

Antioxidant activities of essential oils from aromatic plants are mainly attributed to the active compounds present in them. This can be due to the high percentage of main constituents, but also to the presence of other

Croat. Chem. Acta 79 (4) 545¿552 (2006)

548

TABLE I. Percentage compositions of twelve essential oils

O. POLITEO et al.

Peak area / %

No. Compound

RI (a)

HP-20M Clove Coriander Basil Mint pepper Laurel Marjoram Everlast Nutmeg Fennel Cinnamon Sage

Black

HP-101 /

1

Salvene

825 / –

0.5

2

a

-Thujene

930 / 1032

2.2

1.8

3

a

-Pinene

936 / 1038

1.1

2.2

3.3

1.9

11.3

15.8

0.2

4.5

4

Camphene

954 / 1060

2.8

5

Sabinene

975 / 1092

9.5

1.2

3.6

25.4

6

b

-Pinene

972 / 1102

0.2

0.9

0.6

13.4

1.5

7

D

3 -Carene

1009 / 1131

1.3

0.4

8

Myrcene

981 / 1148

0.8

0.8

0.6

2.0

0.5

9

a

-Phellandrene

978 / 1161

0.4

0.8

1.0

10

a

-Terpinene

996 / 1163

0.3

1.4

0.3

11.0

0.4

2.0

11

1,8-Cineole

1027 / 1179

3.5

3.8

34.9

14.1

12

Limonene

1023 / 1183

0.2

8.8

0.9

0.3

4.6

3.4

0.6

13

b -Phellandrene

1001 / 1187

3.4

1.7

14

g -Terpinene

1049 / 1231

1.6

0.7

0.6

0.8

16.3

0.8

3.9

15

p -Cymene

1020 / 1247

0.8

0.2

0.2

1.5

0.4

0.7

16

a -Terpinolene

1083 / 1260

1.3

0.2

2.8

0.1

1.0

17

( Z )-2-methyl- 2-butene acid

1028

/ 1272

0.4

18

Dodecane

1190 / 1329

0.2

19

Fenchone

1062 / 1365

12.4

20

( E )-2-methyl- 2-butene acid

1128

/ 1373

1.6

21

4-methyl anisole

– / 1392

0.2

22

Thujone

1104 / 1396

56.5

23

trans -Sabinene

/ 1431

1.1

hydrate

 

24

Isomenthone

1029 / 1452

30.9

25

a -Copaene

1365 / 1466

0.5

4.2

0.4

0.9

26

g -Elemene

1482 / 1469

2.4

0.2

27

Camphor

1122 / 1482

1.4

0.6

0.3

5.7

28

b -Bourbonene

1354 / –

0.1

29

Linalool

1085 / 1507

92.0

23.5

13.5

3.7

0.7

0.3

30

Menth-8-ene

– / 1530

3.7

31

Bornyl acetate

1266 / 1550

0.4

0.3

0.2

0.3

0.3

32

Neoisomenthol

1145 / 1556

3.6

33

Terpinen-4-ol

1162 / 1561

2.4

40.8

6.2

t

34

b -Elemene

1364 / –

0.6

35

Dihydrocarvone

1169 / –

0.4

36

Caryophyllene

1395 / 1585

1.2

0.6

2.6

57.6

2.1

1.3

6.7

0.7

0.9

37

Germacrene B

1400 / 1606

3.4

38

Neomenthol

1153 / 1612

44.1

39

Alloaroma–

1447 / 1613

0.2

0.2

dendrene

 

40

trans -Pino–

– / 1614

0.1

carveole

 

41

a

-Terpineol

1295 / 1624

0.2

1.2

0.3

0.3

6.1

0.5

0.4

0.2

42

a

-Humulene

1430 / 1638

0.1

0.4

0.2

2.6

0.3

0.6

6.9

43

Fenchol

– / 1646

1.0

Croat. Chem. Acta 79 (4) 545¿552 (2006)

(cont.)

ANTIOXIDANT ACTIVITY OF SPICE ESSENTIAL OILS

549

(cont.)

Peak area / %

No. Compound

RI (a)

HP-20M Clove Coriander Basil Mint pepper Laurel Marjoram Everlast Nutmeg Fennel Cinnamon Sage

Black

HP-101 /

44 Estragole

1183 / 1655

24.7

2.2

45 g -Cadinene

1428 / –

0.4

46 Borneol

1165 / –

0.7

47 Germacrene D

1442 / 1669

0.5

1.2

0.3

48 Piperitone

1216 / 1673

0.6

49 a

-Cedrene

– / 1674

18.3

50 a

-Muurolene

1506 / 1683

0.3

0.3

51 Carvone

– / 1684

0.8

0.3

52 Neryl acetate

1345 / 1692

0.3

7.6

53 b

-Farnesene

1452 / –

0.3

54 b

-Bisabolene

1499 / 1694

0.2

4.6

55 b - Selinene

1419 / 1695

1.3

3.4

56 Benzenepropanal

1140 / 1709

0.3

57 D

-Cadinene

1497 / 1716

t

0.2

0.2

0.3

0.2

1.9

0.2

0.4

58 a

-Zingiberene

– / 1724

1.3

59 a

-Farnesene

1518 / 1725

0.4

0.6

0.3

60 ar-Curcumene

– / 1747

5.1

61 Nerol

1223 / 1762

1.2

62 a -Bergamotene

1414 / 1779

2.7

0.8

0.2

63 Geraniol

– / 1787

1.0

64 2-Methylcyclohex-

/ 1798

10.5

yl pentanoate

65 trans -Anethole

1273 / 1809

0.2

0.1

77.6

66 Safrole

– / 1809

3.3

67 Cresole

– / 1812

1.4

68 2-Methylcyclohex-

/ 1856

2.1

yl octanoate

69 a -Terpinyl

1333

/ 1880

12.2

acetate

70 Methyl

1281 / 1900

1.5

cinnamate (b)

71 Caryophyllene

– / 1917

2.3

oxide

72 cis -Calamenene

1549 / 1927

0.3

0.2

73 a

-Amorphene

1439 / –

2.3

0.1

74 a

-Guaiene

1404 / –

0.2

75 Methyl eugenol

1390 / 1947

4.1

13.5

0.9

76 Anisaldehyde

– / 1947

0.6

77 Geranyl

1421

/ 1956

0.2

propanoate

78 Nerolidol

1513 / 1996

0.2

79 trans -Cinnam

1280

/ 1997

94.0

aldehyde

80 Neryl

1685

/ 2017

0.2

propionate

81 Methyl

1364

/ 2020

11.1

cinnamate (b)

82 1567 / 2083

Guaiol

0.4

0.3

(cont.)

Croat. Chem. Acta 79 (4) 545¿552 (2006)

550

O. POLITEO et al.

(cont.)

Peak area / %

No. Compound

RI (a)

HP-20M Clove Coriander Basil Mint pepper Laurel Marjoram Everlast Nutmeg Fennel Cinnamon Sage

Black

HP-101 /

83 a -Cadinol

– / 2085

4.4

0.2

84 Eugenol

1377 / 2098

91.2

0.5

11.6

3.4

0.2

85 Eugenyl acetate

– / 2107

7.4

86 Torreiol

– / 2112

0.4

87 Thymol

1362 / 2115

0.2

0.2

0.2

0.5

88 Elemicine

1521 / 2165

0.3

89 b - Eudesmol

1613 / 2176

0.3

90 g -Gurjunene

1616 / –

0.2

91 Myristicine

1496 / –

14.8

92 Chavicol

– / c

0.6

93 Coumarin

c / –

0.3

Total:

99.9

98.6

97.6 97.4

97.6

93.0

96.3

92.8

99.5

93.9

98.4

95.3

(a)

(b)

(c)

RI, retention indices relative to C 8 -C 22 alkanes on polar HP-20 M and apolar HP-101 columns (sorted according to HP-20 M)

Correct isomer is not identified

Retention times are outside retention times of homologous series of C 8 -C 22 alkanes (identified by MS)

t Peak area < 0.1%

- Not identified

constituents in small quantities or to synergy among them. 32 In this study, the antioxidant activities related to the contents of essential oils of twelve aromatic spice plants belonging to different plant families were deter- mined. The results are summarized in Table II. It was found that the essential oils of all analyzed plants sho- wed very different antioxidant capacities. Stronger activ- ity is indicated by a higher antioxidant index determined by each of the three different methods: DPPH, FRAP

and TBARS. In contrast, the RANCIMAT test showed almost the same results for all tested oils. The results from Table II suggest that the essential oils from three spice plants, i.e., clove, basil and laurel, could be used as a potential source of natural antioxidants with possible applications in food systems. The antioxidant activity of clove essential oil is mainly due to the high content of eugenol. The same result was previously indicated by the lipid-malonaldehyde assay. 33

TABLE II. Antioxidant activity of twelve essential oils using the corresponding concentrations (A = 50 g/L , B = 20 g/L, C = 10 g/L, D = 5 g/L) measured by four different methods: DPPH, FRAP, TBARS and RANCIMAT

 

DPPH

 

FRAP

TBARS

 

RANCIMAT

% inhibition

mmol / L

 

AI %

AAI

 

A

B

C

D

A

B

C

D

A

B

CD

A

Clove

94

93

93

93

740

440

131

88

65

49

32

22

1.5

Basil

93

93

88

85

78

25

13

7

45

29

26

22

1.1

Laurel

93

89

80

68

22

10

4

2

38

18

4

1

1.1

Coriander

88

69

44

30

12

5

2

<1

39

21

18

9

1.0

Nutmeg

82

56

39

24

11

3

2

<1

67

40

30

24

1.1

Black Pepper

61

37

22

14

11